The present invention relates to chemical analysis and, more particularly, to an improved method for capillary electrophoresis. A major objective of the present invention is to provide for more convenient and precise determinations of the absolute concentration of a sample component in a sample solution.
Chemical analyses of complex organic structures has made noteworthy advances in biotechnology possible. Biotechnology has provided techniques for manufacturing life-supporting medicines and other products which would otherwise be in short supply if natural sources had to be relied upon. In addition, entirely new medical products are in development which may arrest and cure heretofore untreatable diseases. Biotechnology promises new products for agriculture which will feed the world's expanding populations and which will enhance the ability of famine-prone countries to sustain themselves.
Chemical analysis of biological samples generally involves the separation of the samples into components for identification and quantification. Capillary zone electrophoresis (CZE) is one of a class of methods in which the different components are moved within a narrowbore capillary at respective and different rates so that the components are divided into distinct zones. The distinct zones can be investigated within the capillary or outside the capillary by allowing the components to emerge from the capillary for sequential detection.
In CZE, a sample is introduced at an input end of a longitudinally extending capillary and moved toward an output end. Electrodes of different potentials at either end of the capillary generate the electrical forces which move the sample components toward the output end of the capillary. This movement includes two distinct components, one due to electro-osmotic flow and the other due to electrophoretic migration.
One of the applications of CZE is to determine the absolute concentration of a sample component in a sample solution. The quantity of a sample component eluting from a separation column can be determined by integrating its detection peak to determine the area represented by the component.
However, the quantity of sample solution from which the component quantity was separated is not easily ascertained. The problems of obtaining precise sample volume introductions have been studied in "Theory, Instrumentation, and Applications of Capillary Zone Electrophoresis" by Krynn DeArman Lukacs, a dissertation submitted in 1983 to the University of Carolina at Chapel Hill. This dissertation examined electrostatic introduction, using the same mechanism used by CZE for sample separation and concluded that precise volume control was not obtainable due to differential electrophoretic mobility of sample components. Various hydrostatic introductions were made, basically using syringes, but convective disturbances and parabolic flow profiles adversely affected the sample distribution in the column. The sample should be in the form of a compact cylindrical plug at the column head. Other shapes and distributions result in broader eluting bands and, thus, poorer detection sensitivity.
Attempts to determine sample quantities from the detected peaks have had limited success. It is not feasible to determine a total sample quantity by integrating over all eluting peaks. It is possible to introduce a known quantity of a identifiable component into a sample solution and use the area of its peak to determine the volume separated. However, the introduction of the identifiable component into the sample solution must be carefully controlled if useful results are to be obtained. More problematic is the selection of the identifiable component which must have a peak which will not interfere with the sample component peaks. It is often necessary to run a sample to find detection regions without peaks to select the sample. This is undesirably cumbersome.
What is needed is a convenient and reliable method for determine absolute concentrations of sample components.